The precise regulation of the events occurring during embryonic development as well as during tissue repair in adult organ systems is modulated in part by transcription factors.
Certain disease states, such as Dilated Cardiomyopathy (DCM), have been linked to inappropriate transcriptional regulation. DCM is a leading cause of cardiovascular morbidity and mortality and is characterized as a heterogeneous group of myocardial diseases characterized by cardiac dilation and impaired myocardial contractility (Richardson, P. et al (1996) Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies. Circulation 93:841-842). This syndrome consists of ventricular enlargement, abnormal systolic and diastolic left ventricular function, symptoms of congestive heart failure, and premature death due predominantly to heart failure and cardiac arrhythmias. Coronary artery disease, valvular heart disease, viral infection, toxins, autoimmunity, and primary genetic abnormalities can all cause dilated cardiomyopathy, but in many patients it is idiopathic (Leiden, J. M. (1997) N Engl J Med 337:1080-1081). Studies have indicated that a common set of molecular and cellular pathways accounts for the progression of this disease.
To date, two classes of genes have been implicated in DCM. The first class comprises genes that encode structural proteins like dystrophin (Muntoni, F. et al (1993) N Engl. J Med 329:921-925) and muscle LIM (Lin-11, Isl-1, and Mec-3) protein (Arber, S. et al (1997) Cell 88:393-403.; Arber, S. et al (1994) Cell 79:221-231). These proteins organize the contractile apparatus of cardiac myocytes and ensure their structural integrity. A related disease, Marfan's syndrome, also effects the cellular-extracellular relationship in the heart. Marfan's syndrome is an autosomal dominant disorder of connective tissue that is characterized by ocular, skeletal, and cardiovascular manifestations. With a combination of diligent tracking of the cardiovascular status of Marfan's patients, prophylactic aortic-root replacement, and the use of beta-adenergic-blocking agents morbidity and mortality from cardiovascular failure has decreased. The effective treatment of patients with Marfan's syndrome relies on early and accurate diagnosis. Heretofore, there has been a lack of sensitive and specific diagnostic tests for the disorder. A cause-and-effect relationship has been determined between mutations in the fibrillin gene (a glycoprotein component of the extracellular microfibril) and the Marfan's phenotype (Dietz, H. C. et al (1991) Nature 352:337-339).
A second class of genes, those which encode transcription factors that control the expression of cardiac myoctye genes, have also been implicated in DCM. For example, the cyclic AMP response-element binding protein (CREB) is a basic leucine-zipper nuclear transcription factor that regulates the expression of genes in response to a wide variety of extracellular signals. A dominant-negative CREB mouse model revealed a four chambered DCM phenotype closely resembling many of the anatomical, physiological, and clinical features of human Idiopathic-Dilated Cardiomyopathy (IDC) wherein monocyte numbers decreased, interstitial fibrosis occurred and impaired systolic and diastolic left ventricular function was in evidence (Fentzke R. C. et al (1998) J Clin Invest 101(11):2415-2426) Expression of certain "fetal" genes, which are normally repressed after embryonic development, is a common feature in cardiac hypertrophy. A transcription factor that has been implicated in cardiac function and specifically in the developmental progression of cardiac organogenesis is nuclear factor of activated T cells (NF-ATc). Studies with NF-ATc nonsense-mutation mouse models reveal that NF-ATc is required for the proper development of the pulmonary and aortic valves and septum in the heart. (de la Pompa, J. L. et al (1998) Nature 392:182-186.; Ranger, A. M. (1998) Nature 392: 186-190) NF-ATc, having translocated to the nucleus via a calcineurin mediated pathway, may be able to form a complex with a developmentally expressed transcription factor, GATA4, to activate so-called fetal genes (Molkentin, J. D. et al (1998) Cell 93 (2): 215-28). Geneticists have identified five additional loci associated with adult-onset autosomal dominant dilated cardiomyopathy. Soon it will be possible to correlate clinical outcome with genetic susceptibility profiles, as has been reported for patients with hypertrophic cardiomyopathy.
The immune system is a highly regulated and plastic system with a variety of stimulatory and responsive elements. One modality for the regulation of stimulus response and the subsequent exquisitely controlled response is via transcription factors which act on a variety of genes in the immune system singularly and in concert with one another. One example of such a transcription factor is nuclear factor-(kappa)B (NF-.kappa.B). This factor regulates the expression of many of the genes involved in proinflammatory pathways such as cytokines, chemokines, enzymes involved in mediating inflammation, immune receptors and adhesion molecules involved in the initial recruitment of leukocytes to sites of inflammation (Stein, B, and Baldwin, A. S. (1993) Mol Cell Biol 13:7191-7198; Kopp, E. B. and Ghosh, S. (1995) Adv Immunol 58:1-27). It plays a role in asthma, ulcerative colitis and rheumatoid arthritis by regulating the expression of the inducible gene for nitric oxide synthase (Xie, Q. W. et al (1994) J Biol Chem 269:4705-4708) and it modulates the onset of inflammatory disease via the regulation of cyclooxygenase-2 increasing the production of prostaglandins and thromboxanes (Yamamoto, K. et al (1995) J Biol Chem 270:31315-50; Crofford, L. J. et al (1994) J Clin Invest 93:1095-101). Changes in the expression or activation of specific oncogenes encoding transcription factors cause many leukemias characterized by particular chromosomal translocations (Rabbitts, T. H. (1994) Nature 372:143-9.). T-cell acute leukemias may have a variety of genes fused to their T-cell-receptor gene loci, but the fusion partners have a common function: they are almost all genes for transcription factors (Fisch, P. et al (1992) Oncogene 7:2389-97; Korsmeyer, S. J. (1992) Annu Rev Immunol 10:785-807; Cleary, M. L. (1991) Cell 66:619-22; Cline, M. J. (1996) N Engl J Med 330:328-336), for example, in acute childhood leukemia the expression of the homeobox-containing gene HOX-11 is activated by translocation to the T-cell receptor locus (Hatano, M. et al (1991) Science 253:79-82). The molecular characterization of the defects associated with diseases such as are stated herein point the way towards therapeutic approaches. Immunosupressive agents such as cyclosporin and tacrolimus (FK 506) exert their effects by inhibiting specific transcription factors that are required for T-cell activation (Liu, J. et al (1991) Cell 66:807-15). Thus, it is clear that a greater understanding of role which transcription factors play in the immune system would lead to the determination of highly specific drug targets which would work to treat immune system disorders, such as chronic inflammatory disease.
Other embryonic developmental transcription factors play integral roles in organogenesis and tissue repair. A subset of these factors, called T-Box transcription factors, share several common features: DNA-binding and transcriptional regulatory activity; retention of conserved expression patterns between orthologs and within subfamilies; modulation of regulatory pathways; mediation of mesodermal induction as well as other inductive interactions; and some modulate embryogenesis, organogenesis, organ regeneration, and tissue repair.
The mouse Brachyury (T) gene was the first T-Box gene to be discovered (Dobrovolskaia-Zavadskaia, N. (1927) C.R. Seanc Soc Biol 97:114-116.) and it is by far the most studied. Recently it was identified by positional cloning (Herrmann et al. (1990) Nature 343:617-622.) and was found to be a murine semi-dominant mutation that caused a short tail in heterozygotes, and embryonic lethality in homozygotes. The T-protein was described as having a highly conserved DNA-binding domain known as a T-Box (Pflugfelder et al. (1992) Biochem Biophys Res Commun 186:918-925; Bollag et al. (1994) Nat Genet 7:383-389). This DNA-binding domain binds a 24 base pair palindromic element (AATTTC ACACCT AGGTGT GAAATT) and regulates transcription though two pairs of activation and repression domains (Kispert et al. (1995) EMBO J 14:4763-4772).
Sequence homology was found between the mouse T gene and a cloned Drosophila gene called omb (Pflugfelder et al., Biochem Biophys Res Commun 186:918-925, 1992). The Xenopus Brachyury (Xbra) induces different mesodermal cell types in a dose-dependent manner. (O'Reilly et al. (1995) Development 121:1351-1359). Expression of Xbra in Xenopus is an immediate-early response to mesoderm-inducing factors, such as members of the transforming growth factor-.beta. (TGF-.beta.) family and the fibroblast growth factor (FGF) family (as reviewed by Smith et al. (1995) Semin Dev Biol 6:405-410).
There is a high level of conservation associated with this isolated region of each member of the T-Box family. The T-Box extends across a region of 180 to 190 amino acid residues, which can be located at any position within the polypeptide (Agulnik, S. I., et al. (1996) Genetics 144:249-254; Agulnik et al. (1997) Genome 40: 458-464). Thus far, no sequence similarity has been found outside the T-Box region among different T-Box family members.
The T-Box gene family can be said to consist of several generic entities: T, Tbr-1, Tbx1-9, 11, 12, 17 and T2 and many species has been shown to contain orthologs. Several mouse T-Box genes have been reported; mu-T, mu-Thr1 (identified in a subtractive hybridization screen for genes specifically involved in regulating forebrain development (Bulfone et al. (1995) Neuron 15:63-78), mu-Tbx1-6, mm-Tbx13 (Wattler et al., Genomics 48:24-33), and mm-Tbx14 (Wattler et al. (1998) Genomics 48:24-33, 1998). There are four Xenopus genes (Xbra, x-eomes, x-ET and x-VegT (Zhang et al. (1996) Development 122:4119-4129; Smith et al. (1995) Semin Dev Biol 6:405-410; Lustig et al. (1996) Development 122:4001-4012; Stennard et al. (1996) Development 122:4179-4188; Horb et al. (1997) Development 124:1689-1698; Ryan et al. (1996) Cell 87:989-1000).
Human orthologs for six of eight mouse genes have been identified. Hu-T (Edwards et al. (1996) Genome Res 6:226-233; Morrison et al. (1996) Hum Mol Genet 5:669-674) and hu-TBR1 (Bulfone et al. (1995) Neuron 15:63-78) were found by homology with the mouse orthologs. Hu-TBX2 was isolated independently by two groups from embryonic kidney cDNA libraries (Campbell et al. (1995) Genomics 28:255-260; Law et al. (1995) Mamm Genome 6:267-277). Hu-TBX1, hu-TBX3, and hu-TBX5 were found during investigations aimed at uncovering the genetic basis of human developmental dysmorphic syndromes and were recognized as orthologs of the mouse genes by sequence homology (Li et al. (1997) Nat Genet 15:21-29; Basson et al. (1997) Nat Genet 15:30-35; Chieffo et al. (1997) Genome 43:267-277).
There is currently only a handful of known mutations in T-Box genes. Spontaneous mutations in hu-TBX3 (Bamshad et al. (1997) Nat Genet 16:311-315) and hu-TBX5 (Li et al. (1997) Nat Genet 15:21-29; Basson et al. (1997) Nat Genet 15:30-35) have been reported. These mutations at T-Box genes play a role in several human autosomal, dominant developmental syndromes: Ulnar-Mammary syndrome and Holt-Oram syndrome. Ulnar-Mammary syndrome is characterized by limb defects, abnormalities of apocrine glands such as the absence of breasts, axillary hair and perspiration, dental abnormalities such as ectopic, hypoplastic and absent canine teeth, and genital abnormalities such as micropenis, shawl scrotum and imperforate hymen. Holt-Oram syndrome is characterized by cardiac septal defects and preaxial radial ray abnormalities of the forelimbs (Li et al. (1997) Nat Genet 15:21-29; Basson et al. (1997) Nat Genet 15:30-35; Bamshad et al. (1997) Nat Genet 16:311-315). Mutations in the 5' end of TBX5 lead to substantial cardiovascular malformations and relatively mild skeletal defects while mutations in the 3' end of the gene cause severe skeletal malformation and have less effect on cardiac development (McCarthy, M (1998) Lancet 351(9115):1564; Basson, C. T. et al (1997) Nature Genetics 15:30-35).
A better understanding of the role which T-Box transcription factors play in embryogenesis, organogenesis and organ regeneration has been recently recognized. T-Box related genes have been found in many species, making up a large group of T-Box transcription factors which are highly conserved in their DNA-binding capacity but may be highly divergent in the non-DNA-binding regions. There are common features which define the family, as well as specific differences that define individual members. Phylogenetic analysis suggests that the genome of most animal species will have at least five T-Box genes (related to mu-Tbx2, mu-Tbx, mu-Tbx1, mu-T, and mu-Thr1). There are at least 16 distinct members in 11 different animal groups that have been reported and human orthologs of six of the eight mouse genes have already been identified. The human orthologs of the other mouse T-Box genes have yet to be revealed.
Given the importance of such T-Box DNA-binding transcription factors in proper embryogenesis, organogenesis, organ regeneration and tissue repair, there exists a need to identify other novel transcription factors which function to regulate cell differentiation, whose aberrant function can result in developmental disorders such as Ulnar-Mammary syndrome and Holt-Oram syndrome, and which can be used in the treatment of organ injury by way of regeneration and/or tissue repair such as in hibernating myocardium during myocardial ischemia. By identifying the genes that initiate and exacerbate dilated cardiomyopathy, and by assembling the gene products into biochemical pathways, therapeutic targets for new drugs and gene therapies for this disease may be discovered.